Study Of HIF-1α On The Expression Of RANKL In Osteoblasts Induced By Hypoxia And Salidroside On The Regulation Of HIF-1α Pathway In Osteoclasts Under Hypoxic Conditions

The imbalance in bone remodeling is due to the disruption of the coupling between osteoblast-mediated bone formation and osteoclast-mediated bone resorption,resulting in various diseases of abnormal bone metabolism.More and more studies have shown that hypoxia is closely related to the occurrence of osteoporosis.A considerable number of people in our country live in a plateau chronic hypoxic environment.Therefore,it is very important to study the effect of hypoxia on bone metabolism.Hypoxia is an important regulator of bone metabolism.HIF-1 is a core transcription factor that induces hypoxia genes and repairs the intracellular microenvironment.It can promote the transcription of hypoxia-responsive genes,activate the transcription of downstream target genes,and trigger cells series of adaptive responses to hypoxia.It has been confirmed that hypoxia can cause massive accumulation of HIF-1α,which in turn activates osteoclast activity,promotes differentiation and increases bone resorption activity.RANKL is a key factors that regulate osteoclast differentiation and function.It is secreted by osteoblasts.Hypoxia or endogenous overexpression of HIF-1αcan promote the expression of RANKL,which can be induced by binding to the RANK receptor on the surface of osteoclasts.Salidroside（SAL）is the main active ingredient in Rhodiola,with anti-hypoxia,anti-cancer,anti-depression,protection of central nervous system,protection of cardiovascular system and other pharmacological activities.In our early research confirmed that SAL can mediate angiogenesis and bone formation coupling by activating osteoblast HIF-1α/VEGF pathway under hypoxic conditions,so as to improve bone density,bone biomechanics and morphometric indexes of ovariectomized rats.Whether oxygen-induced osteoblast HIF-1αcan indirectly promote osteoclast differentiation and function by directly up-regulating its own expression of RANKL,and whether the effect of SAL on osteoclasts also affects osteoclasts by activating the oxygen-sensing HIF-1 signaling pathway,there is no report on differentiation and function.In this study,based on literature reports and previous work,the use of mouse osteoclast precursor RAW264.7 cells,mouse bone marrow-derived macrophages（BMMs）,human osteoblast-like MG-63 cells,mouse osteoblast-like MC3T3-E1 cell model,to study the role of HIF-1αon hypoxia-induced osteoblast RANKL expression at the cellular and molecular level,and the regulation of SAL on osteoclast HIF-1αpathway under hypoxia,for the future clinical application of SAL prevention and treatment provide a basis for osteoporosis.Methods:1 Human osteoblast-like MG-63 cells and mouse osteoblast-like MC3T3-E1 cells were selected as research models.The cells were treated in two ways:hypoxia（1%O₂）and simulated hypoxia（CoCl₂）.The experiments were divided into three groups:（1）Control group;（2）1%O₂group;（3）CoCl₂ group（500μM）,using q PCR and ELISA experimental techniques to detect the effect of hypoxia on RANKL gene and protein expression in MG-63 cells and MC3T3-E1cells.Further application of the HIF-1αinhibitor YC-1 confirmed that the above-mentioned effect of hypoxia on RANKL expression was mediated by HIF-1α.The method was the same as above,except that the cells were pretreated with YC-1（10μM）or DMSO for 1 h before hypoxia,and a YC-1 control group was established.2 Using liposome transient transfection technology,introduce ss HIF-1αand its empty vector p CMVh-HA or HIF-1αsh RNA and scramble sh RNA into osteoblasts respectively,and detect endogenous overexpression and inhibition by Western Blot,ELISA,q PCR technology the effect of HIF-1αon the gene and protein expression levels of RANKL in osteoblasts.3 The liposome method was used to co-transfect p GL3-h RANKL promoter-luc plasmid with ss HIF-1αor p CMVh-HA and SV40-βgal into osteoblasts.After 24 h,hypoxia was used for 24 h.Genetic technology was used to detect the effect of HIF-1αon the transcriptional activity of osteoblast RANKL.4 Using Ch IP technology to determine whether HIF-1αcan bind to the RANKL promoter gene in osteoblast chromatin under hypoxic conditions.5 Using CoCl₂ hypoxic conditions,the experiment was divided into six groups:（1）Control group;（2）CoCl₂ group（100μM）;（3）CoCl₂+SAL（1 n M）group;（4）CoCl₂+SAL（10 n M）group;（5）CoCl₂+SAL（100 n M）group;（6）CoCl₂+SAL（1000 n M）group;using Western Blot,ELISA,q PCR technology to detect SAL on osteoclast precursor cells RAW264.7 HIF-1α,p VHL,VEGF,ANGPTL4,IL-6 and mi R-20a gene and protein expression.6 Induce RAW264.7 cells with RANKL（50 ng/m L）and M-CSF（25 ng/m L）for 4 days,induce them into mature osteoclasts,and identify by TRAP staining;use the same conditions and methods as in 5 to detect the effects of SAL on the gene and protein expression levels of HIF-1α,p VHL,VEGF,ANGPTL4,IL-6 and mi R-20a in mature osteoclast RAW264.7 induced by RANKL and M-CSF.7 Select 4-6 week-old male C57BL/6 mice,take bilateral femoral bone marrow under sterile conditions,extract mouse bone marrow-derived macrophages（BMMs）of mouse bone marrow,use TRAP staining,cell surface antibody detection to identify primary osteoclasts purity;using MTT and Western Blot technology to explore the equivalent of 1%O₂ CoCl₂concentration;and using RANKL（50 ng/m L）and M-CSF（50 ng/m L）to induce BMMs cells for 4 days induced into mature osteoclasts,using the same conditions and methods as in 5 to detect SAL on BMMs HIF-1α,p VHL,VEGF,ANGPTL4,IL-6 and mi R-20a gene and protein expression effects.8 Using immunofluorescence and Western blot technology to observe the effect of SAL on the nuclear translocation of HIF-1αin RAW264.7 cells under hypoxic conditions,and the liposome method to transiently transform mouse p GL3-4×HRE-luc plasmid into RAW264.7cells,luciferase reporter gene technology was used to detect the effect of SAL on the transcriptional activity of HIF-1αin RAW264.7 cells under hypoxic conditions.9 Using YC-1（10μM）to conduct the blocking experiment on the above experiment,and select the concentration with the most obvious SAL effect.The experiment is divided into five groups:（1）Control group;（2）YC-1 group;（3）CoCl₂ group（100μM）;（4）CoCl₂+SAL group;（5）YC-1+CoCl₂+SAL group.To investigate the effects of YC-1 on the gene and protein expression levels of HIF-1α,p VHL,VEGF,ANGPTL4,IL-6 and mi R-20a,and the effects of HIF-1αnuclear translocation and transcriptional activity in RAW264.7 cells under hypoxic conditions.Results:1 Hypoxia（1%O₂）or simulated hypoxia（500μM CoCl₂）can significantly increase the gene and protein expression levels of RANKL in human osteoblast-like MG-63 cells and mouse osteoblast-like MC3T3-E1 cells.Application of HIF-1αspecific blocking agent YC-1 can block the regulation of hypoxia on the expression of RANKL.2 Endogenous overexpression of HIF-1αcan up-regulate the gene and protein expression levels of RANKL in MG-63 cells,while inhibiting endogenous HIF-1αexpression can down-regulate the expression of RANKL,suggesting that oxygen-induced osteoblasts under hypoxic conditions.It is also possible to indirectly promote osteoclast differentiation by inducing HIF-1αto directly upregulate its own RANKL expression.3 Under hypoxic conditions,overexpression of HIF-1αcan significantly up-regulate the transcriptional activity of the RANKL gene promoter in MG-63 cells.4 Hypoxia can promote the binding of HIF-1αto the RANKL promoter gene in the chromatin of MG-63 cells.5 Under hypoxic conditions,SAL promoted the gene and protein expression levels of HIF-1αand its downstream target genes VEGF,ANGPTL4 and IL-6 in RAW264.7 cells,and inhibited the expressions level of its downstream target genes mi R-20a and upstream regulatory genes p VHL.6 Through TRAP detection,RAW264.7 cells induced by RANKL and M-CSF were differentiated into mature osteoclasts.Under hypoxic conditions,SAL could promote the gene and protein expression levels of HIF-1αand its downstream target genes VEGF,ANGPTL4 and IL-6 in RAW264.7 cells induced by RANKL and M-CSF,and inhibit the expressions level of mi R-20a and p VHL.7 Fluorescence detection and TRAP identification of surface antibodies proved that the extracted mouse bone marrow cells were primary osteoclast precursor cells with a purity of97.9%;using MTT and Western Blot technology,the CoCl₂ concentration equivalent to 1%O₂was found 50μM;under hypoxic conditions,SAL can promote the gene and protein expression levels of HIF-1αand its downstream target genes VEGF,ANGPTL4 and IL-6 in BMMs cells,and inhibit the expression of downstream target gene mi R-20a and upstream regulatory gene p VHL.Consistent with the results of RAW264.7 cells.8 Under the hypoxic conditions,SAL could promote the nuclear translocation of HIF-1αin RAW264.7 cells,and the protein quantity of HIF-1αin the cytoplasm and nucleus is increased,and the protein expression in the nucleus increased significantly,and SAL also promotes the transcription activity of HIF-1α.9 YC-1 can significantly inhibit the gene and protein expression levels of HIF-1αand its downstream target genes VEGF,ANGPTL4 and IL-6,and promote the expression level of mi R-20a and p VHL caused by SAL.YC-1 can also inhibit the nuclear translocation and transcriptional activity of HIF-1αinduced by SAL on hypoxic conditions.Conclusions:1 HIF-1αexpression can up-regulate the expression of osteoblast RANKL.HIF-1αpromotes its transcription by binding to the gene promoter of RANKL molecule and then up-regulates its expression.2 Under hypoxic conditions,SAL can promote the expression of downstream target genes VEGF,ANGPTL4 and IL-6 in osteoclast HIF-1αsignaling pathway,and inhibit the expression of downstream target gene mi R-20a and upstream regulatory gene p VHL.3 Under hypoxic conditions,SAL can promote the expression of HIF-1αby promoting the nuclear translocation and increasing the transcription activity of HIF-1αin osteoclasts.